We want to understand, on a molecular level, enzyme catalyses in which oxidation and reduction of prosthetic groups, cofactors, and substrates occur. We wish to relate this knowledge to the functioning of these catalysts in cellular metabolism. We have chosen to study important groups of metalloproteins, with an integrated approach using sensitive and selective techniques such as electron paramagnetic resonance (EPR), ENDOR, absorbance, and Mossbauer spectroscopy in combination with chemical modifications of proteins, chemical determination of metal and cofactor composition, and steady-state and pre-steady state kinetic techniques. We will focus on the following areas of investigation: 1) Simple iron-sulfur proteins (ferredoxins) - We propose to develop chemical and spectroscopic procedures to determine the types and numbers of iron- sulfur clusters in these proteins and to relate structural and function features by comparison of physical and chemical properties to crystallographic structure. 2) Iron-sulfur enzymes - We propose to characterize the iron-sulfur prosthetic groups in (among others) nitrogenase, hydrogenase, TPNH-sulfite reductase, and mitochondrial electron-transfer complexes, and to elucidate electron-transfer pathways and control mechanisms operative in these cases. 3) TPNH-nitrate reductase - We propose to characterize the enzyme from N. crassa, to define the sequence of electron transfers, and to identify themolybdenum cofactor from this enzyme and from nitrogenase. 4) Adrenal steroid hydroxylases - We propose to characterize the cholesterol side chain cleavage cytochrome P450 from bovine adrenal mitochondria, to determine the sequence of hydroxylations that occur, and to study in vivo and in vitro steroidogenesis in the rat, with emphasis on a combination of EPR and optical spectroscopic approaches.